Colorectal cancer (CRC) is the third most common form of cancer and the second leading cause of cancer-related deaths in the western world. Its natural evolution (adenoma-carcinoma sequence is believed to occur in most patients) and accessibility by non-surgical methods makes it suitable for early detection and prevention.
Early detection greatly improves the chances of curing CRC. Colonoscopy is an extremely specific and sensitive test, which could be considered the reference method for an early diagnosis of CRC [44,45], and is typically recommended to be performed at 10-year intervals for detection, usually beginning at the age of 50 [1]. However, it is an invasive test which requires good bowel preparation and the patient's cooperation. It is also associated to a minor risk of serious complications, reducing its generalised use.
Fecal occult blood testing (FOBT) is generally considered to be the best non-invasive test for CRC, since it reduces cancer-related mortality when used programmatically [2]. However, its sensitivity is fairly low (15% for adenomas and 35% for carcinomas), implying that a substantial proportion of colorectal neoplasms are missed [1]. Additionally, it has a low specificity giving rise to a lot of false positives. Also, some tests require patients to change their diet, which considerably reduces its usability [46-47].
In this context, DNA testing in non-invasive samples appears as a feasible approach that can complement and probably outperform other screening test for CRC [3-5] [23-26], although to date, its routine clinical application is still a matter of debate [6-7, 27].
DNA appears to be stable in stool, and although PCR inhibitors are present, purification steps have made it possible to carry out PCR-based analyses systematically in a high proportion of samples [28-30]. This proportion is even higher when lesions, especially colorectal carcinomas, are diagnosed. Stool samples in subjects with CRC can contain more exfoliated cells and/or amplifiable DNA [31].
Most of the studies published to date using stool DNA have detected mutations in single genes or markers (e.g., K-ras, p53, APC or BAT26) [16,26,32-33] with acceptable although variable sensitivity and specificity. Others have postulated the use of a multiple target mutation assay panel, which in a large screening setting, showed a sensitivity of 51.6% for carcinomas and 32.5% for adenomas with high grade dysplasia [29]. These promising results are associated with a moderate reduction in the level of specificity (95%), due to the detection of mutations in endoscopically normal colons.
Methylation of the cytosine residues of CpG-rich sequences (CpG islands), located within the gene promoter regions, is usually associated with gene silencing and is considered as a mechanism for cancer gene inactivation.
The genomes of pre-neoplastic, cancerous and aged cells share three important changes in the methylation levels, as early events in the development of certain tumours. Firstly, the hypomethylation of heterochromatin which leads to genomic instability and increases the events of mitotic recombination; secondly, hypermethylation of individual genes, and finally, hypermethylation of the CpG islands of constitutive genes and tumour suppressant genes. The two levels of methylation can occur individually or simultaneously. In general, hypermethylation is involved in gene silencing and hypomethylation in the over-expression of certain proteins linked to the processes of invasion and metastasis.
Promoter hypermethylation of many genes, such as p16INK4a, p14ARF, APC, MGMT, LKB1, hMLH1, RASSF1A, CRBP1, RARB2 [8-10], are a common and early events in colorectal tumorigenesis.
In the last few years, changes in the methylation of cytosine residues of CpG-rich sequences (CpG islands) have been increasingly recognised as very common molecular aberrations presumed to be of clinical use [34]. In colorectal cancer, methylated markers are interesting for screening because they occur with high frequency in early stage neoplasia [35]. Detection of CpG islands methylation in human DNA isolated from stool [11-12,36] or serum [37-39] has been proposed as a novel strategy for the early diagnosis of colorectal neoplasia. Thus, the methylation status of the gene promoter regions such as p16, MGMT, MLH1, SFRP2, HIC1 and vimentine in stool DNA has been evaluated, either individually or using a combined panel [11-15,25,40, 48-53]. To date, the methylation of SFRP2 is the most sensitive individual marker (sensitivity of 77-90%). However, both individual and combined methylated markers have been hampered by a relatively high proportion of false positives.
Assesment of promoter hypermethylation in stool DNA could provide a valuable approach to the non-invasively screening for early colorectal lesions [11-15]. Notwithstanding, the lack of specificity demonstrated until now made its potential clinical use difficult. The DNA resulting from the exfoliation of the colonic epithelium in the faecal matter is difficult to amplify given the enormous number of PCR inhibitors found in stools.
Based on the current needs and difficulties in establishing a non invasive early diagnosis of colorectal cancer with acceptable sensitivity and specificity, the authors of the invention have developed a panel of biomarkers of methylation in stool suitable for routine use in clinical practice.
Thus, following considerable research, the authors have found a panel of 5 methylated markers (RARB2, MGMT, p16INK4a, APC and p14ARF) which jointly represent most tumours.
After finding these five markers, the authors of the invention, have developed a new method for the early diagnosis of colorectal cancer based on the analysis of the melting curves through real-time PCR detection (RT PCR Melting curve, MC) of the state of methylation of the promoters of the 5 genes: RARB2, p16INK4a, MGMT, p14ARF and APC in stool DNA, demonstrating high sensitivity in the early detection of CRC tumours and excellent specificity (no false positives in normal colonoscopies).
This new method allows the non invasive detection of pre-malignant and malignant neoplastic alterations of the colon with high sensitivity and excellent specificity, allowing its routine use in clinical practice.